Tag: Gottschalk

Dysregulation of tetrahydrobiopterin metabolism in myalgic encephalomyelitis/chronic fatigue syndrome by pentose phosphate pathway

Abstract:

Background: Tetrahydrobiopterin (BH4) and its oxidized derivative dihydrobiopterin (BH2) were found to be strongly elevated in ME/CFS patients with orthostatic intolerance (ME + OI).

Objective: However, the molecular mechanism of biopterin biogenesis is poorly understood in ME + OI subjects. Here, we report that the activation of the non-oxidative pentose phosphate pathway (PPP) plays a critical role in the biogenesis of biopterins (BH4 and BH2) in ME + OI subjects.

Research design and results: Microarray-based gene screening followed by real-time PCR-based validation, ELISA assay, and finally enzyme kinetic studies of glucose-6-phosphate dehydrogenase (G6PDH), transaldolase (TALDO1), and transketolase (TK) enzymes revealed that the augmentation of anaerobic PPP is critical in the regulations of biopterins. To further investigate, we devised a novel cell culture strategy to induce non-oxidative PPP by treating human microglial cells with ribose-5-phosphate (R5P) under a hypoxic condition of 85%N2/10%CO2/5%O2 followed by the analysis of biopterin metabolism via ELISA, immunoblot, and dual immunocytochemical analyses. Moreover, the siRNA knocking down of the taldo1 gene strongly inhibited the bioavailability of phosphoribosyl pyrophosphate (PRPP), reduced the expressions of purine biosynthetic enzymes, attenuated GTP cyclohydrolase 1 (GTPCH1), and suppressed subsequent production of BH4 and its metabolic conversion to BH2 in R5P-treated and hypoxia-induced C20 human microglia cells. These results confirmed that the activation of non-oxidative PPP is indeed required for the upregulation of both BH4 and BH2 via the purine biosynthetic pathway. To test the functional role of ME + OI plasma-derived biopterins, exogenously added plasma samples of ME + OI plasma with high BH4 upregulated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in human microglial cells indicating that the non-oxidative PPP-induced-biopterins could stimulate inflammatory response in ME + OI patients.

Conclusion: Taken together, our current research highlights that the induction of non-oxidative PPP regulates the biogenesis of biopterins contributing to ME/CFS pathogenesis.

Source: Bulbule S, Gottschalk CG, Drosen ME, Peterson D, Arnold LA, Roy A. Dysregulation of tetrahydrobiopterin metabolism in myalgic encephalomyelitis/chronic fatigue syndrome by pentose phosphate pathway. J Cent Nerv Syst Dis. 2024 Aug 19;16:11795735241271675. doi: 10.1177/11795735241271675. PMID: 39161795; PMCID: PMC11331476. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11331476/ (Full text)

Augmentation of Anaerobic Pentose Phosphate Pathway Dysregulates Tetrahydrobiopterin Metabolism in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) Patients with Orthostatic Intolerance: A Pilot Study

Abstract:

Tetrahydrobiopterin (BH4), an essential cofactor of amino acid metabolism, was found to be strongly elevated in ME/CFS patients with Orthostatic intolerance (ME + OI). However, the molecular mechanism of BH4 upregulation is poorly understood in ME + OI patients. Here, we report that the activation of the non-oxidative pentose phosphate pathway (PPP) plays a critical role in the biosynthesis of BH4 in ME + OI patients.

Microarray-based gene screening followed by real-time PCR-based validation, ELISA assay, and finally enzyme kinetic studies of glucose-6-phosphate dehydrogenase (G6PDH), transaldolase (TALDO1), and transketolase (TK) enzymes revealed that the augmentation of anaerobic PPP is critical in the pathogenesis of ME + OI. Along with the upregulated anaerobic PPP enzymes, we observed that biopterin metabolites such as BH4 and dihydrobiopterin (BH2) are strongly upregulated suggesting the disruption of biopterin homeostasis in ME + OI patients.

To explore the molecular role of anaerobic PPP in biopterin metabolism, we devised a novel cell culture strategy to induce non-oxidative PPP by treating human microglial cells with ribose-5-phosphate (R5P) under a hypoxic condition of 85%N2/10%CO2/5%O2 followed by the analysis of BH4 and BH2 upregulation via ELISA, immunoblot and dual immunocytochemical analyses.

These results confirmed that the activation of non-oxidative PPP is indeed required for the upregulation of both BH4 and BH2. Moreover, the siRNA knocking down of the taldo1 gene strongly inhibited the expression of GTP cyclohydrolase 1 (GTPCH1) and subsequent production of BH4 and its metabolic conversion to BH2 in R5P-treated and hypoxia-induced C20 human microglia cells. To test the functional role of ME + OI plasma-derived biopterins, exogenously added plasma samples of ME + OI plasma with high BH4 upregulated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in human microglial cells indicating that the non-oxidative PPP-induced-biopterins could stimulate inflammatory response in ME + OI patients.

Source: Sarojini Bulbule, Carl Gunnar Gottschalk, Molly E Drosen et al. Augmentation of Anaerobic Pentose Phosphate Pathway Dysregulates Tetrahydrobiopterin Metabolism in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) Patients with Orthostatic Intolerance: A Pilot Study, 11 December 2023, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-3716093/v1] https://www.researchsquare.com/article/rs-3716093/v1 (Full text)

Altered Fatty Acid Oxidation in Lymphocyte Populations of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Abstract:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a disabling multisystem illness in which individuals are plagued with fatigue, inflammatory symptoms, cognitive dysfunction, and the hallmark symptom, post-exertional malaise. While the cause of this disease remains unknown, there is evidence of a potential infectious component that, along with patient symptoms and common onsets of the disease, implicates immune system dysfunction. To further our understanding of the state of ME/CFS lymphocytes, we characterized the role of fatty acids in isolated Natural Killer cells, CD4+ T cells, and CD8+ T cells in circulation and after overnight stimulation, through implicit perturbations to fatty acid oxidation.

We examined samples obtained from at least 8 and as many as 20 subjects for immune cell fatty acid characterization in a variety of experiments and found that all three isolated cell types increased their utilization of lipids and levels of pertinent proteins involved in this metabolic pathway in ME/CFS samples, particularly during higher energy demands and activation. In T cells, we characterized the cell populations contributing to these metabolic shifts, which included CD4+ memory cells, CD4+ effector cells, CD8+ naïve cells, and CD8+ memory cells.

We also discovered that patients with ME/CFS and healthy control samples had significant correlations between measurements of CD4+ T cell fatty acid metabolism and demographic data. These findings provide support for metabolic dysfunction in ME/CFS immune cells. We further hypothesize about the consequences that these altered fuel dependencies may have on T and NK cell effector function, which may shed light on the illness’s mechanism of action.

Source: Maya J, Leddy SM, Gottschalk CG, Peterson DL, Hanson MR. Altered Fatty Acid Oxidation in Lymphocyte Populations of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Int J Mol Sci. 2023 Jan 19;24(3):2010. doi: 10.3390/ijms24032010. PMID: 36768336; PMCID: PMC9916395. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9916395/ (Full text)

Potential molecular mechanisms of chronic fatigue in long haul COVID and other viral diseases

Abstract:

Historically, COVID-19 emerges as one of the most devastating diseases of humankind, which creates an unmanageable health crisis worldwide. Until now, this disease costs millions of lives and continues to paralyze human civilization’s economy and social growth, leaving an enduring damage that will take an exceptionally long time to repair.

While a majority of infected patients survive after mild to moderate reactions after two to six weeks, a growing population of patients suffers for months with severe and prolonged symptoms of fatigue, depression, and anxiety. These patients are no less than 10% of total COVID-19 infected individuals with distinctive chronic clinical symptomatology, collectively termed post-acute sequelae of COVID-19 (PASC) or more commonly long-haul COVID. Interestingly, Long-haul COVID and many debilitating viral diseases display a similar range of clinical symptoms of muscle fatigue, dizziness, depression, and chronic inflammation.

In our current hypothesis-driven review article, we attempt to discuss the molecular mechanism of muscle fatigue in long-haul COVID, and other viral diseases as caused by HHV6, Powassan, Epstein-Barr virus (EBV), and HIV. We also discuss the pathological resemblance of virus-triggered muscle fatigue with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).

Source: Gottschalk CG, Peterson D, Armstrong J, Knox K, Roy A. Potential molecular mechanisms of chronic fatigue in long haul COVID and other viral diseases. Infect Agent Cancer. 2023 Feb 7;18(1):7. doi: 10.1186/s13027-023-00485-z. PMID: 36750846; PMCID: PMC9902840. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9902840/ (Full text)

Elevated ATG13 in serum of patients with ME/CFS stimulates oxidative stress response in microglial cells via activation of receptor for advanced glycation end products (RAGE)

Abstract:

Myalgic Encephalomyelitis, also known as Chronic Fatigue Syndrome (ME/CFS), is a multisystem illness characterized by extreme muscle fatigue associated with pain, neurocognitive impairment, and chronic inflammation. Despite intense investigation, the molecular mechanism of this disease is still unknown. Here we demonstrate that autophagy-related protein ATG13 is strongly upregulated in the serum of ME/CFS patients, indicative of impairment in the metabolic events of autophagy.

A Thioflavin T-based protein aggregation assay, array screening for autophagy-related factors, densitometric analyses, and confirmation with ELISA revealed that the level of ATG13 was strongly elevated in serum samples of ME/CFS patients compared to age-matched controls. Moreover, our microglia-based oxidative stress response experiments indicated that serum samples of ME/CFS patients evoke the production of reactive oxygen species (ROS) and nitric oxide in human HMC3 microglial cells, whereas neutralization of ATG13 strongly diminishes the production of ROS and NO, suggesting that ATG13 plays a role in the observed stress response in microglial cells. Finally, an in vitro ligand binding assay provided evidence that ATG13 employs the Receptor for Advanced Glycation End-products (RAGE) to stimulate ROS in microglial cells.

Collectively, our results suggest that an impairment of autophagy following the release of ATG13 into serum could be a pathological signal in ME/CFS.

Source: Gottschalk G, Peterson D, Knox K, Maynard M, Whelan RJ, Roy A. Elevated ATG13 in serum of patients with ME/CFS stimulates oxidative stress response in microglial cells via activation of receptor for advanced glycation end products (RAGE). Mol Cell Neurosci. 2022 Apr 26:103731. doi: 10.1016/j.mcn.2022.103731. Epub ahead of print. PMID: 35487443. https://www.sciencedirect.com/science/article/abs/pii/S1044743122000379?via%3Dihub (Full text)